This investigation will examine the covalent structures of the amino acyl-tRNA synthetases; it will concentrate on a representative of this family of enzymes, isoleucyl-tRNA synthetase of E. coli. Two specific aims will be attacked. First, the amino acid sequence of this enzyme will be determined. Limited proteolytic cleavage of the protein by trypsin and chymotrypsin will be exploited as means of producing polypeptide fragments of sizes amenable to conventional methods of amino acid sequence determination. Second, chemical modifications of the protein will be conducted as a means of identifying functional groups essential for catalysis and for binding substrates. These chemical modification studies will concentrate upon identifying residues already known to react with specific reagents. They will examine functional group specific reagents, such as N-ethyl maleimide which reacts with a cysteinyl residue which is essential for catalysis. They will also consider affinity labeling reagents such as bromoacetyl isoleucyl-tRNA. It is hoped that these studies will identify catalytic residues, lead to an insight on the enzyme mechanism, shed light on the nature of protein-nucleic acid interactions, and give an insight into the mode of evolution of this family of enzymes.